Purification and Characterization of Colicin V from Escherichia coli Culture Supernatants

Abstract

The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa. The protein identified remains biologically active after purification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues. The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC. N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids. The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase. The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria. Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.

Root-hair endophyte stacking in finger millet creates a physicochemical barrier to trap the fungal pathogen Fusarium graminearum

“…Tn5 inserted in a relatively uncharacterized gene required for colicin V production (ewvC) orthologous to cvpA in Escherichia coli 42 . Colicin V is a secreted peptide antibiotic 43 . Consistent with the gene annotation, ewvC-4B9::Tn5 failed to inhibit an E. coli strain that is sensitive to colicin V compared to wild type (Fig.…”

Topology analysis of the colicin V export protein CvaA in Escherichia coli

“…In addition, in contrast to the C-terminal secretion signals of ␣-hemolysin (34) and the E. chrysanthemi proteases (11), the export signal in CvaC has been localized to the N-terminal region (23). It is noteworthy that CvaC has actually been identified as a member of a class of peptide bacteriocins with double-glycine-type leader peptides, which were found previously only in gram-positive bacteria (18,29). Furthermore, like other ABC proteins that mediate export of related bacteriocins, the colicin V transporter CvaB has an N-terminal extension proposed to have leader peptidase activity (28).…”

“…The determinants for colicin V activity, immunity, and export are encoded on low-copy-number virulence plasmids. cvaC encodes the 103-amino-acid toxin precursor; the precursor contains a 15-amino-acid leader peptide of the double-glycine type, which is removed concomitantly with export (18,29). The cvi gene confers immunity to the producing cell, and cvaA and cvaB encode two dedicated export proteins (22).…”

Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11

“…Some bacteriocins require the complementary action of two different peptides to achieve biological activity; this two-peptide group includes lactacin F (2), lactococcin G (39), and most probably lactococcin M (59) as well. The primary translation product of most nonlantibiotics, some lantibiotics, and colicin V contains a leader peptide of double-glycine type which might serve as a recognition signal for protein export (13,17). Such precursor peptides are processed and secreted by a dedicated export system made up by an ABC transporter and its accessory protein (16).…”